ERK-derived peptides and uses thereof

ABSTRACT

An isolated peptide being no longer than 20 amino acids comprising a sequence at least 95% homologous to the sequence GQLNHILGILGX1PX2QED (SEQ ID NO: 4), wherein X1 and X2 are any amino acid, the peptide being capable of preventing extracellular signal-regulated kinase1/2 (ERK) translocation into the nucleus.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.15/022,280 filed on Mar. 16, 2016, which is a National Phase of PCTPatent Application No. PCT/IL2014/050822 having International FilingDate of Sep. 15, 2014, which claims the benefit of priority under 35 USC§ 119(e) of U.S. Provisional Patent Application No. 61/878,633 filed onSep. 17, 2013. The contents of the above applications are allincorporated by reference as if fully set forth herein in theirentirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 69737SequenceListing.txt, created on Apr. 26,2017, comprising 3,047 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to peptidesderived from extracellular signal-regulated kinase1/2 (ERK) which may beused for treating cancer.

The extracellular signal-regulated kinase1/2 (ERK) cascade is anintracellular signaling pathway that regulates cellular processes, suchas proliferation and differentiation. Being a central signalingcomponent, its dysregulation is involved in various pathologies,particularly cancer. Indeed, inhibitors of both Rafs and MEK1/2 withinthe cascade were recently developed, but despite the widespreadinvolvement of ERK in the induction and maintenance of cancers, theseinhibitors were proven beneficial almost only in B-Raf mutatedmelanomas. In addition, most sensitive melanomas develop resistance tothe Raf/MEK inhibitors within several months of treatment. The lack ofeffect in many cancer types, and the mechanisms of acquired resistanceare now being investigated, and shown to often involve the inhibition ofERK-dependent negative feedback loops. Consequently, this inhibitionallows hyperactivation of upstream signaling components that circumventthe inhibited ERK cascade. Hence, inhibiting the ERK cascade withoutaffecting the feedback loops should result in a more general anti cancerdrug.

One of the key steps in the transmission of extracellular signals is thenuclear translocation of ERK. In resting cells, most of ERK is localizedin the cytoplasm due to anchoring to cytoplasmic proteins, butstimulation causes a rapid and massive nuclear translocation of a largeportion of the ERK molecules. The molecular mechanism of translocationinvolves first TEY-phosphorylation-dependent conformational change,which results in the detachment of the ERK molecules from their anchors.This exposes the ERK to an additional phosphorylation on two Serresidues within a nuclear translocation signal (NTS). Thephosphorylation of the NTS then allows the beta-like importin (Imp),Imp7, to bind it, and consequently, induce the nuclear translocation ofthe kinases. This rapid translocation allows the phosphorylation andactivation of many nuclear proteins, which are important for theinduction and regulation of cellular processes.

U.S. Patent Application Publication No. 20100099627 teaches 18 aminoacid peptides that are capable of preventing ERK translocation into thenucleus.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an isolated peptide being no longer than 20 aminoacids comprising a sequence at least 95% homologous to the sequenceGQLNHILGILGX₁PX₂QED (SEQ ID NO: 4), wherein X₁ and X₂ are any aminoacid, the peptide being capable of preventing extracellularsignal-regulated kinase1/2 (ERK) translocation into the nucleus.

According to an aspect of some embodiments of the present inventionthere is provided an isolated peptide being 17 amino acids comprisingthe sequence GQLNHILGILGX₁PX₂QED (SEQ ID NO: 4), wherein X₁ and X₂ areany amino acid, the peptide being capable of preventing ERKtranslocation into the nucleus.

According to an aspect of some embodiments of the present inventionthere is provided a composition of matter comprising the isolatedpeptide described herein, attached to a cell penetrating agent.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof comprising administering to the subject a therapeuticallyeffective amount of the peptide described herein, thereby treating thecancer.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof comprising administering to the subject a therapeuticallyeffective amount of the composition of matter described herein, therebytreating the cancer.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the peptidedescribed herein as an active agent and a pharmaceutically acceptablecarrier.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising thecomposition of matter described herein as the active agents and apharmaceutically acceptable carrier.

According to some embodiments of the invention, the isolated peptide is17 amino acids long.

According to some embodiments of the invention, X₁ and X₂ are eachindependently selected from the group consisting of glutamic acid,aspartic acid, alanine and serine.

According to some embodiments of the invention, X₁ and X₂ are eachindependently selected from the group consisting of glutamic acid andaspartic acid.

According to some embodiments of the invention, X₁ and X₂ are glutamicacid.

According to some embodiments of the invention, X₁ and X₂ are asparticacid.

According to some embodiments of the invention, the isolated peptide isdevoid of the amino acid sequence Leu-Aspartic acid.

According to some embodiments of the invention, neither X₁ nor X₂ isalanine.

According to some embodiments of the invention, the cell penetratingagent comprises myristic acid.

According to some embodiments of the invention, the myristic acid isattached to the N terminus of the peptide.

According to some embodiments of the invention, the cell penetratingagent is a cell penetrating peptide.

According to some embodiments of the invention, the cell penetratingpeptide comprises an amino acid sequence which is attached to the Nterminus of the isolated peptide described herein.

According to some embodiments of the invention, the cell penetratingpeptide comprises an acid sequence as set forth in SEQ ID NO: 5.

According to some embodiments of the invention, the isolated peptidedescribed herein is attached to the cell penetrating peptide via apeptide bond.

According to some embodiments of the invention, the isolated peptideconsists of the amino acid sequence selected from the group consistingof SEQ ID NOs: 1-3, 6 and 7.

According to some embodiments of the invention, the isolated peptideattached to the cell penetrating peptide is no longer than 30 aminoacids.

According to some embodiments of the invention, the peptide is attachedto a cell penetrating agent.

According to some embodiments of the invention, the cancer is selectedfrom the group consisting of melanoma, breast cancer, lung cancer,prostate cancer and cervical cancer.

According to some embodiments of the invention, the cancer is melanoma.

According to some embodiments of the invention, the melanoma comprisesB-Raf melanoma.

According to some embodiments of the invention, isolated peptide is foruse in treating cancer.

According to some embodiments of the invention, the composition ofmatter is for use in treating cancer.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-1F illustrate that EPE peptide prevents nuclear translocationof ERK1. A2352 (A), MDA-MB-231 (B) HeLa (C), T47D (D), NHEM-AD (E) andHB2 (F) cells were serum-starved, pretreated with the EPE peptides, Scrpeptide (10 μM, 2 h) DMSO and then either stimulated with TPA (250 nM,15 min) or left untreated (NS) as control. The cells were stained withαgERK1/2 Ab and DAPI. Scale-bar—15 μm.

FIGS. 2A-2F illustrate that the EPE peptide prevents ERK1/2's NTSphosphorylation, the interaction of ERK1 with importin7 and activationof nuclear ERK1/2 targets. (A) EPE peptide prevents ERK1/2-importin7interaction. HeLa cells were serum starved (0.1%, 16 hours), pretreatedwith EPE peptide, scrambled peptide (10 μM, 2 hours) or DMSO, and thenstimulated with TPA (250 nM, 15 min). ERK1 were precipitated with αgERK1Ab; importin7 co-immunoprecipitated with ERK1 was detected by WB withαimportin7 Ab. (B) EPE peptide prevents ERK1/2's NTS and Elk1 but notRSK1 phosphorylation. T47D, HeLa, MDA-MB-231 and A2352 cells were serumstarved (0.1%, 16 hours), pretreated with EPE peptide, scrambled peptide(10 μM, 2 hours) or DMSO control, and then stimulated with TPA (250 nM,indicated times). Cell extracts were subjected to Western blot analysiswith the indicated Abs. (C) EPE peptide inhibits phosphorylation ofc-Myc and Elk1 in B-Raf melanomas. A2158 and A2352 cells were treatedwith scrambled and EPE peptide for two hours and then stimulated withthe indicated concentration of TPA for 30 min. Cell extracts weresubjected to Western blot analysis with the indicated Abs. (D)Quantification of some of the results in B and C. The bars representfold difference between scrambled and EPE peptides in thephosphorylation of the indicated proteins after TPA stimulation. Theresults are the average of two or three experiments. (E) EPE-peptidedoes not affect short term AKT phosphorylation. The same extractsdescribed in B were subjected to Western blot analysis using anti pAKTand gAKT Abs. (F) The EPE peptide does not affect long-term AKTphosphorylation. HeLa cells were treated with the EPE, or scrambled,peptide (10 μM) or DMSO control for the indicated times after which theywere subjected to Western blot analysis using anti pAKT and gAKT Abs.

FIGS. 3A-3B illustrate that EPE peptide prevents proliferation of cancerbut not normal cells. (A) Effect of the EPE peptide on the proliferationof various cell lines. Sixteen cancer and four “normal” immortalizedcell lines were treated with either EPE or scrambled peptides asdescribed above. Viable cells were assayed by Methylene Blue at 48 or 72hours after cell seeding. The data from at least three independentexperiments is presented as percent of the amount of viablecells/initial cell number, where initial cell number was considered as100. (B) Comparison of the effects of the EPE-peptide and Raf inhibitorPLX4032 on cell viability. NHEM-Ad, LOXIMVI, A2352, Hela MDA-MB-231, andDU-145 cells were treated with EPE peptide, scrambled peptide, (10 μM),PLX4032 (1 μM), DMSO control, or no treatment at 1% FCS. The number ofcells was detected as above. The graphs present the kinetic of cellgrowth at the indicated times. All experiments were repeated 3 times intriplicates.

FIGS. 4A-4B illustrate that the EPE peptide prevents proliferation ofresistant melanoma cells. (A) A2352 melanoma cells resistant to eitherPLX4032 (B-Raf) or U0126 (MEK) inhibitors were treated with DMSO, EPE orScr peptide (10 μM), U0126 (10 μM), PLX4032 (1 μM), Wortmannin (0.5 μM)or Taxol (25 μg/ml). Presented as percent of viable cells at 72h/initial cell number. (B) Resistant melanomas from patients. Twovemurafenib-resistant melanoma lines, A4132 and A4168, were grown in 1%FCS and treated with medium containing DMSO, EPE peptide (10 μM),PLX4032 (1 μM), PD-184352 (5 μM) or combined treatment of EPE peptide(10 μM) plus PLX4032 (1 μM), administrating fresh medium every 24 hoursduring the 96 hours experiment. Methylene blue assay was performed toquantify viable cells. Experiments were done twice in triplicate. Datapresented as fold change. (*) p<0.01, (#) p<0.05 with respect to theDMSO control.

FIGS. 5A-5D illustrate that the EPE peptide leads to apoptosis of B-Rafmutant cancer cells. (A) The EPE peptide affects the morphology oftreated cells. The indicated cells were treated with either the EPE orScr peptides, DMSO, or taxol for 72 h, and photographed by a lightmicroscope. (B) The EPE peptide induces apoptosis in melanoma cellsdetected by TUNEL. The indicated cell lines were treated as in A for 24h. Apoptosis was detected by TUNEL staining. Images were obtained byflorescence microscope. (C) Quantification of B. The bar-graphrepresents percent of apoptotic cells calculated in 10 different fields.(D) The EPE peptide induces apoptosis of melanoma cells detected by PARPcleavage. The indicated cell lines were treated as in (A) for 24 h. Cellextracts were subjected to Western blotting with αPARP Ab.

FIGS. 6A-6B illustrate that the EPE peptide prevents the growth of humantumor xenografts. CD-1 nude mice were inoculated with MDA-MB-231,LOXIMVI and HCT-116 cells, and SKID mice were inoculated with A2352cells. Upon tumors establishment, the mice were treated intravenouslywith the DMSO as well as EPE or Scr peptides in the indicated doses, 3times a week. The results are averages of 5 mice in each experimentalgroup. The X, Y, Z dimensions of the tumors were measured with acaliper, and the volume was calculated accordingly. The results areaverages of 5 mice in each experimental group. The significance of allexperiments was at least P<0.001. (B) The EPE peptide prevents tumorrecurrence on melanoma xenograft. SCID mice were inoculated with A2352cells. Upon establishment of tumors mice were treated intravenously with15 mg/kg EPE peptide (n=7) or intraperitonealy with 15 mg/kg PLX4032(n=5), at the indicated dose 3 times a week. Tumor size was recorded atthe same time using a caliper. Following 3 weeks of treatment, mice werekept for further evaluation monitoring for any tumor recurrence of theeffective treated melanoma xenografts. Experiment was concluded 110 dayspost inoculation (11 weeks after the last treatment) and the overallstate of the animals was evaluated. sac=sacrificed, recur=recurrence,met=metastasis.

FIGS. 7A-7B illustrate the intracellular distribution of the NTS-derivedpeptide. HeLa cells were serum starved, and treated for the indicatedtimes with SPS peptide conjugated with biotin on its C terminus andeither TAT (FIG. 7B) or myristic acid (FIG. 7A) on its N terminus. Theintracellular distribution of the peptide was visualized usingfluorescent microscope. Scale bar 15 μm.

FIGS. 8A-8B illustrate that the EPE peptide does not affect JNK or p38translocation into the nucleus. (A) The EPE peptide does not affect thenuclear translocation of JNK2 and p38. HeLa cells were serum-starved,pretreated with the EPE or Scr peptides, and then stimulated with eitherTPA (250 nM, 15 min), anisomycin (Anis, 10 μg/ml, 15 min) for theindicated times, or left untreated as control (NT). The cells werestained with αp38 or αgJNK Abs and DAPI. Scale bar—15 μm. (B) Extractsfrom cells treated as in A were analyzed by Western blotting with theindicated Abs.

FIGS. 9A-9B illustrate the dose response of the EPE peptides on melanomacells proliferation. LOXIMVI (FIG. 9A) and A2352 (FIG. 9B) melanomacells were treated with the indicated doses of the EPE or Scr peptides.Presented as percent of viable cells at 72 h/initial cell number.

FIGS. 10A-10B illustrate that the EPE peptide causes cytoplasmaticlocalization of ERK in xenografts. FIG. 10A: Photographs illustratingthat EPE and Scr peptide as well as DMSO-treated MDA-MB-231 and LOXIMVIxenografts were immunostained with αgERK Ab. FIG. 10B presentsbar-graphs representing number of cells with cytoplasmatic/nuclear ERKstaining calculated in 10 random microscopic fields.

FIG. 11 illustrates that nuclear translocation of ERK1/2 is inhibited byNTS-derived peptides.

FIGS. 12A-12D illustrate that the EPE peptide inhibits the nucleartranslocation of ERK1/2. Serum-starved (0.1% FCS, 16 hr) A2352 (FIG.12A), HeLa (FIG. 12B), MDA-MB-231 (FIG. 12C), or LOXMVI (FIG. 12D) cellswere pretreated with EPE or scrambled peptides (10 μM, 2 hr). Cells werethen stimulated with EGF (50 ng/ml) or left untreated, and thenharvested. Subcellular fractionation of cytoplasm and nucleus wasperformed as below, and fractions were subjected to Western blotanalysis with the indicated Abs. Subcellular Fractionation was performedas follows: Harvested cells were resuspended in 200 μl of buffer Hcontaining 0.1% Nonidet P-40. The lysates were mixed vigorously andcentrifuged immediately to yield supernatants containing the cytosolicfraction. Nuclear proteins were extracted by resuspending the nuclearpellets in 200 μl of extraction buffer, waiting on ice for 5 min, briefsonication (2×5 sec, 40 W, 4° C.), vigorous mixing, and centrifugation.Both cytosolic and nuclear fractions were subjected to Western blotting.

FIGS. 13A-13C illustrate that the EPE peptide has a similar effect onthe nuclear translocation of both ERK1 and ERK2. A2185 (A) and A2352 (B)B-Raf melanoma cells were serum-starved (16 hours, 0.1% FBS), and theneither pretreated with the EPE peptides (10 μM, 2 hours) or leftuntreated. Then, the cells were stimulated with TPA (100 μM, 20 min), orleft untreated (NS) DMSO as control. The cells were stained with eitheranti ERK1 (Santa Cruz, C16) or anti ERK2 (C14, Santa Cruz) Abs and DAPI.(C) Quantification of the number of cell in which ERK1 or ERK2 weremostly (>80%) localized in the nucleus was performed by counting 5fields containing 100 cells per field.

FIGS. 14A-14B illustrate that the EPE peptide reduces the expression andphosphorylation of c-FOS in A2352 cells. (A) Effect of the EPE peptideon c-Fos. A2352 cells were serum starved (0.1%, 16 hours), pretreatedwith EPE peptide or scrambled peptide (10 μM, 2 hours), and thenstimulated with TPA (50 and 100 nM, 60 min.) or left untreated (NS).Cell extracts were subjected to Western blot analysis with the indicatedAbs. The anti g-c-FOS and p-c-FOS Abs were purchased from Santa Cruz(Calif.). (B) Quantification of the results in the 100 nM TPA in A.

FIGS. 15A-15B illustrate that the EPE peptide prevents nuclearaccumulation/translocation of ERK1/2 in PLX4032-resistant melanomacells. A2352 that were made resistant to PLX4032 (as described underMaterial and Methods) and A4132 PLX4032 resistant melanoma cell frompatient were serum-starved (16 hours, 0.1%), pretreated with the EPE orscrambled peptide (10 μM, 2 hours) and then either stimulated with TPA(100 nM, 20 min) or left untreated (NT) as control. The cells werestained with αgERK1/2 Ab and DAPI.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to peptidesderived from extracellular signal-regulated kinase1/2 (ERK) which may beused for treating cancer.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

ERK1/2 signaling plays a crucial role in the induction of proliferation,as well as cancer development and progression. Inhibitors of the ERKcascade (e.g. vemurafenib and trametinib) serve as anti-cancer drugs.However, the majority of these agents have only a limited effect onhuman malignancies, and even the most effective inhibitors affect only alimited number of cancers. In addition, these inhibitors may haveserious side effects including the induction of skin cancer, and thetreated cancers (i.e. melanoma) develop resistance to the drugs within6-8 months. Much of the shortcomings of the current inhibitors areprobably mediated by reduced negative feedback loops.

The present inventors sought to prevent the nuclear translocation ofERK1/2, thus preventing ERK-dependent proliferation but not the negativefeedback loops induced by it. The present inventors synthesized numerouspeptides based on the nuclear translocation signal of ERK and showedthat they were able to efficiently and specifically inhibit theinteraction of ERK with Imp7, thereby preventing the nucleartranslocation of ERK, without changing AKT activity that is usuallyenhanced by inhibition of the ERK-related negative feedback loops.

The EPE based peptide was shown to inhibit the stimulated nucleartranslocation of ERK in all the cell lines examined; however, its effecton cell proliferation varied in different cell lines. The most notableeffect of the peptide was on B-Raf melanoma cells, which underwentapoptosis a few hours following treatment (FIGS. 5A-5D).

The application of the peptide to cultured cells induced apoptosis ofmelanoma cells, while inhibiting the proliferation/survival of othercancer cells, including PLX4032 and U0126-resistant melanoma cells(FIGS. 4A-4B); however, it had no effect on the proliferation ofimmortalized cells (FIGS. 3A-3B). When used in xenograft models,systemic application of the EPE peptide inhibited the growth of breast,colon and melanoma-derived tumors, and eradicated the growth oflow-passage melanoma xenografts (FIGS. 6A-6B and 10A-10B).

Thus, according to one aspect of the present invention there is providedan isolated peptide being no longer than 20 amino acids comprising asequence at least 95% homologous to the sequence GQLNHILGILGX₁PX₂QED(SEQ ID NO: 4), wherein X₁ and X₂ are any amino acid, the peptide beingcapable of preventing extracellular signal-regulated kinase1/2 (ERK)translocation into the nucleus.

The phrase “being capable of preventing extracellular signal-regulatedkinase1/2 (ERK) translocation into the nucleus” refers to the ability todown-regulate the amount of ERK from translocating from the cytoplasminto the nucleus by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more. Methods of detecting whether a peptide is capable of preventingERK translocation are described in the Examples section, herein below.

The peptide of this aspect of the present invention may be 16, 17, 18,19 or 20 amino acids long.

The peptide has an amino acid sequence which is typically at least 94%homologous or identical to the sequence as set forth in SEQ ID NO: 4,95% homologous or identical to the sequence as set forth in SEQ ID NO:4, 96% homologous or identical to the sequence as set forth in SEQ IDNO: 4, at least 97% homologous or identical to the sequence as set forthin SEQ ID NO: 4, at least 98% homologous or identical to the sequence asset forth in SEQ ID NO: 4, at least 99% homologous or identical to thesequence as set forth in SEQ ID NO: 4 or 100% homologous or identical tothe sequence as set forth in SEQ ID NO: 4 as determined using theStandard protein-protein BLAST [blastp] software of the NCBI.

As mentioned, X₁ and X₂ in SEQ ID NO: 4 may be any amino acid (asspecified herein below). According to one embodiment, X₁ and X₂ are eachindependently selected from the group consisting of glutamic acid,aspartic acid, alanine and serine. For example, the X₁ and X₂ may bothbe glutamic acid. For example, the X₁ and X₂ may both be aspartic acid.For example, the X₁ and X₂ may both be serine. For example, X₁ may beglutamic acid and X₂ may be aspartic acid or X₁ may be aspartic acid andX₂ may be glutamic acid. According to another embodiment, neither X₁ norX₂ is alanine.

Thus, according to this aspect of the present invention the peptide isat least 94% homologous or identical to the sequence as set forth in SEQID NO: 2, 95% homologous or identical to the sequence as set forth inSEQ ID NO: 2, 96% homologous or identical to the sequence as set forthin SEQ ID NO: 2, at least 97% homologous or identical to the sequence asset forth in SEQ ID NO: 2, at least 98% homologous or identical to thesequence as set forth in SEQ ID NO: 2, at least 99% homologous oridentical to the sequence as set forth in SEQ ID NO: 2 or 100%homologous or identical to the sequence as set forth in SEQ ID NO: 2 asdetermined using the Standard protein-protein BLAST [blastp] software ofthe NCBI, wherein the serine in position X₁ and X₂ is not replaceable byanother amino acid.

Thus, according to this aspect of the present invention the peptide isat least 94% homologous or identical to the sequence as set forth in SEQID NO: 3, 95% homologous or identical to the sequence as set forth inSEQ ID NO: 3, 96% homologous or identical to the sequence as set forthin SEQ ID NO: 3, at least 97% homologous or identical to the sequence asset forth in SEQ ID NO: 3, at least 98% homologous or identical to thesequence as set forth in SEQ ID NO: 3, at least 99% homologous oridentical to the sequence as set forth in SEQ ID NO: 3 or 100%homologous or identical to the sequence as set forth in SEQ ID NO: 3 asdetermined using the Standard protein-protein BLAST [blastp] software ofthe NCBI, wherein the alanine in position X₁ and X₂ is not replaceableby another amino acid.

Thus, according to this aspect of the present invention the peptide isat least 94% homologous or identical to the sequence as set forth in SEQID NO: 6, 95% homologous or identical to the sequence as set forth inSEQ ID NO: 6, 96% homologous or identical to the sequence as set forthin SEQ ID NO: 6, at least 97% homologous or identical to the sequence asset forth in SEQ ID NO: 6, at least 98% homologous or identical to thesequence as set forth in SEQ ID NO: 6, at least 99% homologous oridentical to the sequence as set forth in SEQ ID NO: 6 or 100%homologous or identical to the sequence as set forth in SEQ ID NO: 6 asdetermined using the Standard protein-protein BLAST [blastp] software ofthe NCBI, wherein the glutamic acid in position X₁ and X₂ is notreplaceable by another amino acid.

Thus, according to this aspect of the present invention the peptide isat least 94% homologous or identical to the sequence as set forth in SEQID NO: 7 (GQLNHILGILGDPDQED, 95% homologous or identical to the sequenceas set forth in SEQ ID NO: 7, 96% homologous or identical to thesequence as set forth in SEQ ID NO: 7, at least 97% homologous oridentical to the sequence as set forth in SEQ ID NO: 7, at least 98%homologous or identical to the sequence as set forth in SEQ ID NO: 7, atleast 99% homologous or identical to the sequence as set forth in SEQ IDNO: 7 or 100% homologous or identical to the sequence as set forth inSEQ ID NO: 7 as determined using the Standard protein-protein BLAST[blastp] software of the NCBI, wherein the aspartic acid in position X₁and X₂ is not replaceable by another amino acid.

Peptides which are not 100% homologous to the sequences disclosed hereinmay comprise either conservative or non-conservative substitutions,deletions or additions.

The term “conservative substitution” as used herein, refers to thereplacement of an amino acid present in the native sequence in thepeptide with a naturally or non-naturally occurring amino or apeptidomimetics having similar steric properties. Where the side-chainof the native amino acid to be replaced is either polar or hydrophobic,the conservative substitution should be with a naturally occurring aminoacid, a non-naturally occurring amino acid or with a peptidomimeticmoiety which is also polar or hydrophobic (in addition to having thesame steric properties as the side-chain of the replaced amino acid).

As naturally occurring amino acids are typically grouped according totheir properties, conservative substitutions by naturally occurringamino acids can be easily determined bearing in mind the fact that inaccordance with the invention replacement of charged amino acids bysterically similar non-charged amino acids are considered conservativesubstitutions.

For producing conservative substitutions by non-naturally occurringamino acids it is also possible to use amino acid analogs (syntheticamino acids) well known in the art. A peptidomimetic of the naturallyoccurring amino acids is well documented in the literature known to theskilled practitioner.

When effecting conservative substitutions the substituting amino acidshould have the same or a similar functional group in the side chain asthe original amino acid.

The phrase “non-conservative substitutions” as used herein refers toreplacement of the amino acid as present in the parent sequence byanother naturally or non-naturally occurring amino acid, havingdifferent electrochemical and/or steric properties. Thus, the side chainof the substituting amino acid can be significantly larger (or smaller)than the side chain of the native amino acid being substituted and/orcan have functional groups with significantly different electronicproperties than the amino acid being substituted. Examples ofnon-conservative substitutions of this type include the substitution ofphenylalanine or cycohexylmethyl glycine for alanine, isoleucine forglycine, or —NH—CH[(—CH₂)₅—COOH]—CO— for aspartic acid. Thosenon-conservative substitutions which fall within the scope of thepresent invention are those which still constitute a polypeptide beingable to prevent ERK translocation into the nucleus.

Preferably, the peptides of the present invention are typically devoidof the sequence Leu-Aspartic acid.

Further, preferably the N terminal amino acid of the peptide is glycine.

The term “peptide” as used herein encompasses native peptides (eitherdegradation products, synthetically synthesized peptides or recombinantpeptides) and peptidomimetics (typically, synthetically synthesizedpeptides), as well as peptoids and semipeptoids which are peptideanalogs, which may have, for example, modifications rendering thepeptides more stable while in a body or more capable of penetrating intocells. Such modifications include, but are not limited to N terminusmodification, C terminus modification, peptide bond modification,backbone modifications, and residue modification. Methods for preparingpeptidomimetic compounds are well known in the art and are specified,for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter17.2, F. Choplin Pergamon Press (1992), which is incorporated byreference as if fully set forth herein. Further details in this respectare provided herein under.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated amide bonds (—N(CH3)-CO—), ester bonds(—C(═O)—O—), ketomethylene bonds (—CO—CH2-), sulfinylmethylene bonds(—S(═O)—CH2-), α-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl (e.g.,methyl), amine bonds (—CH2-NH—), sulfide bonds (—CH2-S—), ethylene bonds(—CH2-CH2-), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), fluorinated olefinic doublebonds (—CF═CH—), retro amide bonds (—NH—CO—), peptide derivatives(—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturally presenton the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) bonds at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted bynon-natural aromatic amino acids such as1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine,ring-methylated derivatives of Phe, halogenated derivatives of Phe orO-methyl-Tyr.

The peptides of some embodiments of the invention may also include oneor more modified amino acids or one or more non-amino acid monomers(e.g. fatty acids, complex carbohydrates etc).

The term “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Tables 1 and 2 below list naturally occurring amino acids (Table 1), andnon-conventional or modified amino acids (e.g., synthetic, Table 2),which can be used with some embodiments of the invention.

TABLE 1 One-letter Three-Letter Symbol Abbreviation Amino Acid A AlaAlanine R Arg Arginine N Asn Asparagine D Asp Aspartic acid C CysCysteine Q Gln Glutamine E Glu Glutamic Acid G Gly Glycine H HisHistidine I Ile Isoleucine L Leu Leucine K Lys Lysine M Met Methionine FPhe Phenylalanine P Pro Proline S Ser Serine T Thr Threonine W TrpTryptophan Y Tyr Tyrosine V Val Valine X Xaa Any amino acid as above

TABLE 2 Non-conventional amino Non-conventional amino Code acid Codeacid Hyp hydroxyproline Orn ornithine Norb aminonorbornyl- Abuα-aminobutyric acid carboxylate Cpro aminocyclopropane- Dala D-alaninecarboxylate Narg N-(3- Darg D-arginine guanidinopropyl)glycine NasnN-(carbamylmethyl)glycine Dasn D-asparagine NaspN-(carboxymethyl)glycine Dasp D-aspartic acid Ncys N-(thiomethyl)glycineDcys D-cysteine Ngln N-(2-carbamylethyl)glycine Dgln D-glutamine NgluN-(2-carboxyethyl)glycine Dglu D-glutamic acid NhisN-(imidazolylethyl)glycine Dhis D-histidine NileN-(1-methylpropyl)glycine Dile D-isoleucine NleuN-(2-methylpropyl)glycine Dleu D-leucine Nlys N-(4-aminobutyl)glycineDlys D-lysine Nmet N-(2-methylthioethyl)glycine Dmet D-methionine NornN-(3-aminopropyl)glycine Dorn D-ornithine Nphe N-benzylglycine DpheD-phenylalanine Nser N-(hydroxymethyl)glycine Dpro D-proline NthrN-(1-hydroxyethyl)glycine Dser D-serine Nhtrp N-(3-indolylethyl) glycineDthr D-threonine Ntyr N-(p-hydroxyphenyl)glycine Dtrp D-tryptophan NvalN-(1-methylethyl)glycine Dtyr D-tyrosine Nmgly N-methylglycine DvalD-valine Nmala L-N-methylalanine Dnmala D-N-methylalanine NmargL-N-methylarginine Dnmarg D-N-methylarginine Nmasn L-N-methylasparagineDnmasn D-N-methylasparagine Nmasp L-N-methylaspartic acid DnmaspD-N-methylasparatate Nmcys L-N-methylcysteine Dnmcys D-N-methylcysteineNmgln L-N-methylglutamine Dnmgln D-N-methylglutamine NmgluL-N-methylglutamic acid Dnmglu D-N-methylglutamate NmhisL-N-methylhistidine Dnmhis D-N-methylhistidine NmileL-N-methylisolleucine Dnmile D-N-methylisoleucine NmleuL-N-methylleucine Dnmleu D-N-methylleucine Nmlys L-N-methyllysine DnmlysD-N-methyllysine Nmmet L-N-methylmethionine Dnmmet D-N-methylmethionineNmorn L-N-methylornithine Dnmorn D-N-methylornithine NmpheL-N-methylphenylalanine Dnmphe D-N-methylphenylalanine NmproL-N-methylproline Dnmpro D-N-methylproline Nmser L-N-methylserine DnmserD-N-methylserine Nmthr L-N-methylthreonine Dnmthr D-N-methylthreonineNmtrp L-N-methyltryptophan Dnmtrp D-N-methyltryptophan NmtyrL-N-methyltyrosine Dnmtyr D-N-methyltyrosine Nmval L-N-methylvalineDnmval D-N-methylvaline Nmnle L-N-methylnorleucine Nle L-norleucineNmnva L-N-methylnorvaline Nva L-norvaline Nmetg L-N-methyl-ethylglycineEtg L-ethylglycine Nmtbug L-N-methyl-t-butylglycine TbugL-t-butylglycine Nmhphe L-N-methyl- Hphe L-homophenylalaninehomophenylalanine Nmanap N-methyl-α-naphthylalanine Anapα-naphthylalanine Nmpen N-methylpenicillamine Pen penicillamine NmgabuN-methyl-γ-aminobutyrate Gabu γ-aminobutyric acid NmchexaN-methyl-cyclohexylalanine Chexa cyclohexylalanine NmcpenN-methyl-cyclopentylalanine Cpen cyclopentylalanine NmaabuN-methyl-α-amino-α- Aabu α-amino-α-methylbutyrate methylbutyrate NmaibN-methyl-α- Aib α-aminoisobutyric acid aminoisobutyrate MargL-α-methylarginine Dmarg D-α-methylarginine Masn L-α-methylasparagineDmasn D-α-methylasparagine Masp L-α-methylaspartate DmaspD-α-methylaspartate Mcys L-α-methylcysteine Dmcys D-α-methylcysteineMgln L-α-methylglutamine Dmgln D-α-methylglutamine MgluL-α-methylglutamate Dmglu D-α-methyl glutamic acid MhisL-α-methylhistidine Dmhis D-α-methylhistidine Mile L-α-methylisoleucineDmile D-α-methylisoleucine Mleu L-α-methylleucine DmleuD-α-methylleucine Mlys L-α-methyllysine Dmlys D-α-methyllysine MmetL-α-methylmethionine Dmmet D-α-methylmethionine Morn L-α-methylornithineDmorn D-α-methylornithine Mphe L-α-methylphenylalanine DmpheD-α-methylphenylalanine Mpro L-α-methylproline Dmpro D-α-methylprolineMser L-α-methylserine Dmser D-α-methylserine Mthr L-α-methylthreonineDmthr D-α-methylthreonine Mtrp L-α-methyltryptophan DmtrpD-α-methyltryptophan Mtyr L-α-methyltyrosine Dmtyr D-α-methyltyrosineMval L-α-methylvaline Dmval D-α-methylvaline Mnva L-α-methylnorvalineNcbut N-cyclobutylglycine Metg L-α-methylethylglycine NchepN-cycloheptylglycine Mtbug L-α-methyl-t-butylglycine NchexN-cyclohexylglycine Mhphe L-α-methyl- Ncdec N-cyclodecylglycinehomophenylalanine Manap α-methyl-α-naphthylalanine NcdodN-cyclododecylglycine Mpen α-methylpenicillamine NcoctN-cyclooctylglycine Mgabu α-methyl-γ-aminobutyrate NcproN-cyclopropylglycine Mchexa α-methyl-cyclohexylalanine NcundN-cycloundecylglycine Mcpen α-methyl-cyclopentylalanine NaegN-(2-aminoethyl)glycine Nnbhm N-(N-(2,2-diphenylethyl) Nbhm N-(2,2-carbamylmethyl-glycine diphenylethyl)glycine NnbheN-(N-(3,3-diphenylpropyl) Nbhe N-(3,3- carbamylmethyl-glycinediphenylpropyl)glycine Tic 1,2,3,4- Nmbc 1-carboxy-1-(2,2-diphenyltetrahydroisoquinoline-3- ethylamino)cyclopropane carboxylic acid pThrphosphothreonine pSer phosphoserine O-methyl-tyrosine pTyrphosphotyrosine hydroxylysine 2-aminoadipic acid

The peptides of some embodiments of the invention are preferablyutilized in a linear form, although it will be appreciated that in caseswhere cyclicization does not severely interfere with peptidecharacteristics, cyclic forms of the peptide can also be utilized.

Since the present peptides are preferably utilized in therapeutics ordiagnostics which require the peptides to be in soluble form, thepeptides of some embodiments of the invention preferably include one ormore non-natural or natural polar amino acids, including but not limitedto serine and threonine which are capable of increasing peptidesolubility due to their hydroxyl-containing side chain.

Further contemplated modifications of the peptides of the presentinvention include C-terminal amidation.

In order to improve the bioavailability of the ERK peptides, a single, aportion or even all the amino acids in the peptide can be D amino acidswhich are not susceptible to enzymatic proteolytic activity and canimprove altogether the use of the peptides of the invention aspharmaceuticals. The peptides of the present invention may be attached(either covalently or non-covalently) to a penetrating agent.

As used herein the phrase “penetrating agent” refers to an agent whichenhances translocation of any of the attached peptide across a cellmembrane.

According to one embodiment, the penetrating agent is a peptide and isattached to the ERK (either directly or non-directly) via a peptidebond. Preferably, the penetrating agent is attached to the N terminus ofthe ERK derived peptide.

Typically, peptide penetrating agents have an amino acid compositioncontaining either a high relative abundance of positively charged aminoacids such as lysine or arginine, or have sequences that contain analternating pattern of polar/charged amino acids and non-polar,hydrophobic amino acids.

By way of a non-limiting example, cell penetrating peptide (CPP)sequences may be used in order to enhance intracellular penetration.CPPs may include short and long versions of the protein transductiondomain (PTD) of HIV TAT protein, such as for example, YARAAARQARA (SEQID NO: 5), YGRKKRR (SEQ ID NO: 8), YGRKKRRQRRR (SEQ ID NO: 9), or RRQRR(SEQ ID NO: 10)]. However, the disclosure is not so limited, and anysuitable penetrating agent may be used, as known by those of skill inthe art.

When the peptides of the present invention are attached to cellpenetrating peptides, it is contemplated that the full length peptide isno greater than 25 amino acids, no greater than 26 amino acids, nogreater than 27 amino acids, no greater than 28 amino acids, no greaterthan 29 amino acids, or no greater than 30 amino acids.

Another method of enhancing cell penetration is via N-terminalmyristoilation. In this protein modification, a myristoyl group (derivedfrom myristic acid) is covalently attached via an amide bond to thealpha-amino group of an N-terminal amino acid of the peptide.

The peptides of some embodiments of the invention may be synthesized byany techniques that are known to those skilled in the art of peptidesynthesis. For solid phase peptide synthesis, a summary of the manytechniques may be found in J. M. Stewart and J. D. Young, Solid PhasePeptide Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J.Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, AcademicPress (New York), 1973. For classical solution synthesis see G. Schroderand K. Lupke, The Peptides, vol. 1, Academic Press (New York), 1965.

In general, these methods comprise the sequential addition of one ormore amino acids or suitably protected amino acids to a growing peptidechain. Normally, either the amino or carboxyl group of the first aminoacid is protected by a suitable protecting group. The protected orderivatized amino acid can then either be attached to an inert solidsupport or utilized in solution by adding the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected, under conditions suitable for forming the amide linkage. Theprotecting group is then removed from this newly added amino acidresidue and the next amino acid (suitably protected) is then added, andso forth. After all the desired amino acids have been linked in theproper sequence, any remaining protecting groups (and any solid support)are removed sequentially or concurrently, to afford the final peptidecompound. By simple modification of this general procedure, it ispossible to add more than one amino acid at a time to a growing chain,for example, by coupling (under conditions which do not racemize chiralcenters) a protected tripeptide with a properly protected dipeptide toform, after deprotection, a pentapeptide and so forth. Furtherdescription of peptide synthesis is disclosed in U.S. Pat. No.6,472,505.

A preferred method of preparing the peptide compounds of someembodiments of the invention involves solid phase peptide synthesis.

Large scale peptide synthesis is described by Andersson Biopolymers2000; 55(3):227-50.

Since the peptides of the present invention are able to specificallyinhibit the nuclear activities of ERK without modulating its cytoplasmicactivities, these peptides may be used to inhibit ERK nuclear activities(e.g. proliferation) without harming other ERK-related cytoplasmicactivities in the cells. Therefore, the peptides of this aspect of thepresent invention may serve as therapeutic agent for hyperproliferativediseases such as cancer without having the side-effects of other ERKinhibitors.

Thus, according to another aspect of the present invention there isprovided a method of treating cancer in a subject in need thereofcomprising administering to the subject a therapeutically effectiveamount of the peptides disclosed herein, thereby treating the cancer.

Examples of cancers that may be treated using the 19S-specificproteasome inhibitors of this aspect of the present invention include,but are not limited to adrenocortical carcinoma, hereditary; bladdercancer; breast cancer; breast cancer, ductal; breast cancer, invasiveintraductal; breast cancer, sporadic; breast cancer, susceptibility to;breast cancer, type 4; breast cancer, type 4; breast cancer-1; breastcancer-3; breast-ovarian cancer; Burkitt's lymphoma; cervical carcinoma;colorectal adenoma; colorectal cancer; colorectal cancer, hereditarynonpolyposis, type 1; colorectal cancer, hereditary nonpolyposis, type2; colorectal cancer, hereditary nonpolyposis, type 3; colorectalcancer, hereditary nonpolyposis, type 6; colorectal cancer, hereditarynonpolyposis, type 7; dermatofibrosarcoma protuberans; endometrialcarcinoma; esophageal cancer; gastric cancer, fibrosarcoma, glioblastomamultiforme; glomus tumors, multiple; hepatoblastoma; hepatocellularcancer; hepatocellular carcinoma; leukemia, acute lymphoblastic;leukemia, acute myeloid; leukemia, acute myeloid, with eosinophilia;leukemia, acute nonlymphocytic; leukemia, chronic myeloid; Li-Fraumenisyndrome; liposarcoma, lung cancer; lung cancer, small cell; lymphoma,non-Hodgkin's; lynch cancer family syndrome II; male germ cell tumor;mast cell leukemia; medullary thyroid; medulloblastoma; melanoma,malignant melanoma, meningioma; multiple endocrine neoplasia; multiplemyeloma, myeloid malignancy, predisposition to; myxosarcoma,neuroblastoma; osteosarcoma; ovarian cancer; ovarian cancer, serous;ovarian carcinoma; ovarian sex cord tumors; pancreatic cancer;pancreatic endocrine tumors; paraganglioma, familial nonchromaffin;pilomatricoma; pituitary tumor, invasive; prostate adenocarcinoma;prostate cancer; renal cell carcinoma, papillary, familial and sporadic;retinoblastoma; rhabdoid predisposition syndrome, familial; rhabdoidtumors; rhabdomyosarcoma; small-cell cancer of lung; soft tissuesarcoma, squamous cell carcinoma, basal cell carcinoma, head and neck;T-cell acute lymphoblastic leukemia; Turcot syndrome with glioblastoma;tylosis with esophageal cancer; uterine cervix carcinoma, Wilms' tumor,type 2; and Wilms' tumor, type 1, and the like.

According to a specific embodiment, the cancer is melanoma, breastcancer, lung cancer, prostate cancer or cervical cancer.

According to another embodiment, the melanoma is PLX4032 and/orU0126-resistant melanoma.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the peptides disclosedherein accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, intraperitoneal, intranasal, orintraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (peptide) effective to prevent, alleviate orameliorate symptoms of a disorder (e.g., cancer) or prolong the survivalof the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer, as furtherdetailed below. Such information can be used to more accuratelydetermine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals, as further detailed below. Thedata obtained from these in vitro and cell culture assays and animalstudies can be used in formulating a range of dosage for use in human.The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to ensure bloodor tissue levels of the active ingredient are sufficient to induce orsuppress the biological effect (minimal effective concentration, MEC).The MEC will vary for each preparation, but can be estimated from invitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

As mentioned, various animal models may be used to test the efficacy ofthe peptides of the present invention. A transgenic mouse model forcancer (e.g., breast cancer) such as the erb model (Shah N., et al.,1999, Cancer Lett. 146: 15-2; Weistein E J., et al., 2000, Mol. Med. 6:4-16) or MTV/myc model (Stewart T A et al., 1984, Cell, 38: 627-637),the c-myc model (Leder A., et al., 1986, Cell, 45:485-495), v-Ha-ras orc-neu model (Elson A and Leder P, 1995, J. Biol. Chem. 270: 26116-22)can be used to test the ability of the peptides of the present inventionto suppress tumor growth in vivo.

For the formation of solid tumors, athymic mice can be injected withhuman or animal (e.g., mouse) cancerous cells such as those derived frombreast cancer, ovarian cancer, prostate cancer or thyroid cancer, andfollowing the formation of cancerous tumors the mice can be subjected tointra-tumor and/or systemic administration of the peptides.

The following cell lines (provided with their ATCC Accession numbers)can be used for each type of cancer model:

For breast cancer:

Human breast cancer cell lines—MDA-MB-453 (ATCC No. HTB-131), MDA-MB-231(ATCC No. HTB-26), BT474 (ATCC No. HTB-20), MCF-7 (ATCC No. HTB-22),MDA-MB-468.

For ovarian cancer:

Human ovarian cancer cell lines—SKOV3 (ATCC No. HTB-77), OVCAR-3HTB-161), OVCAR-4, OVCAR-5, OVCAR-8 and IGROV1;

For prostate cancer:

Human prostate cancer cell lines—DU-145 (ATCC No. HTB-81), PC-3 (ATCCNo. CRL-1435);

For thyroid cancer:

Human derived thyroid cancer cell lines—FTC-133, K1, K2, NPA87, K5,WRO82-1, ARO89-1, DRO81-1;

For lung cancer:

Mouse lung carcinoma LL/2 (LLC1) cells (Lewis lung carcinoma)—Thesecells are derived from a mouse bearing a tumor resulting from animplantation of primary Lewis lung carcinoma. The cells are tumorigenicin C57BL mice, express H-2b antigen and are widely used as a model formetastasis and for studying the mechanisms of cancer chemotherapeuticagents (Bertram J S, et al., 1980, Cancer Lett. 11: 63-73; Sharma S, etal. 1999, J. Immunol. 163: 5020-5028).

For melanoma:

Mouse B16-F10 cells (Melanoma)—The cells are derived from mouse(C57BL/6J) bearing melanoma (Briles E B, et al., 1978, J. Natl. CancerInst. 60: 1217-1222).

The cancerous cells can be cultured in a tissue culture medium such asthe DMEM with 4 mM L-glutamine adjusted to contain 1.5 g/L sodiumbicarbonate and 4.5 g/L glucose, supplemented with 10% fetal calf serum(FCS), according to known procedures (e.g., as described in the ATCCprotocols).

Tumor formation in animal models by administration of cancerouscells—Athymic nu/nu mice (e.g., female mice) can be purchased from theJackson Laboratory (Bar Harbor, Me.). Tumors can be formed bysubcutaneous (s.c.) injection of cancerous cells (e.g., 2×10⁶ cells in100 μl of PBS per mouse). Tumors are then allowed to grow in vivo forseveral days (e.g., 6-14 days) until they reach a detectable size ofabout 0.5 cm in diameter. It will be appreciated that injection ofcancerous cells to an animal model can be at the organ from which thecell line is derived (e.g., mammary tissue for breast cancer, ovary forovarian cancer) or can be injected at an irrelevant tissue such as therear leg of the mouse.

To test the effect of the peptides of the present invention oninhibition of tumor growth, the agents may be administered to the animalmodel bearing the tumor either locally at the site of tumor orsystemically, by intravenous injection of infusion, via, e.g., the tailvein. The time of administration may vary from immediately followinginjection of the cancerous cell line (e.g., by systemic administration)or at predetermined time periods following the appearance of the solidtumor (e.g., to the site of tumor formation, every 3 days for 3-10 timesas described in Ugen K E et al., Cancer Gene Ther. 2006 Jun. 9; [Epubahead of print]).

It will be appreciated that administration may also be effected using anucleic acid construct designed to express the peptide agent (e.g., aviral vector), naked pDNA and/or liposomes.

Tumor sizes are measured two to three times a week. Tumor volumes arecalculated using the length and width of the tumor (in millimeters). Theeffect of the treatment can be evaluated by comparing the tumor volumeusing statistical analyses such as Student's t test. In addition,histological analyses can be performed using markers typical for eachtype of cancer.

According to another embodiment, the agents of the present invention areco-administered or co-formulated with other known chemotherapeuticagents and/or anti-inflammatory agents. In addition, they may beadministered with other known therapies, including but not limited tochemotherapy, radiotherapy, phototherapy and photodynamic therapy,surgery, nutritional therapy, ablative therapy, combined radiotherapyand chemotherapy, brachiotherapy, proton beam therapy, immunotherapy,cellular therapy and photon beam radiosurgical therapy.

Examples of other chemotherapeutic agents which may beco-delivered/coformulated with the agents of the present inventioninclude but are not limited to Acivicin; Aclarubicin; AcodazoleHydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin;Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide;Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; BleomycinSulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide;Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; GemcitabineHydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b;Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Sulofenur; Talisomycin; Taxol; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Tiazofuirin; Tirapazamine; TopotecanHydrochloride; Toremifene Citrate; Trestolone Acetate; TriciribinePhosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride. Additional antineoplastic agents include those disclosedin Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A.Chabner), and the introduction thereto, 1202-1263, of Goodman andGilman's “The Pharmacological Basis of Therapeutics”, Eighth Edition,1990, McGraw-Hill, Inc. (Health Professions Division).

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as is further detailed above.

The term “treating” refers to inhibiting, preventing or arresting thedevelopment of a pathology (disease, disorder or condition) and/orcausing the reduction, remission, or regression of a pathology. Those ofskill in the art will understand that various methodologies and assayscan be used to assess the development of a pathology, and similarly,various methodologies and assays may be used to assess the reduction,remission or regression of a pathology.

As used herein, the term “preventing” refers to keeping a disease,disorder or condition from occurring in a subject who may be at risk forthe disease, but has not yet been diagnosed as having the disease.

As used herein, the term “subject” includes mammals, preferably humanbeings at any age which suffer from the pathology. Preferably, this termencompasses individuals who are at risk to develop the pathology.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Materials and Methods

Peptides:

The 4 peptides used were: (i) Scr—NILSQELPHSGDLQIG (SEQ ID NO: 1); (ii)SPS—GQLNHILGILGSPSQED (SEQ ID NO: 2); (iii) APA—GQLNHILGILGAPAQED (SEQID NO: 3); and (iv) EPE—GQLNHILGILGEPEQED (SEQ ID NO: 6). Each of themwas conjugated in its N-terminal to either a modified TAT sequence(YARAAARQARA²² SEQ ID NO: 5), or myristic acid²³ and C-terminallyamidated. To study the rate of absorption, the SPS peptide wasconjugated to lysine-biotin in their C-terminus. All peptides werepurchased from “Peptide 2” (Chantilly, Va., USA), were more than 85%pure and kept as 100 mM DMSO stock solution at −20 C, until used.

Cells:

HeLa (derived from cervical cancer), MDA-MB-231 (Ras-transformed breastcancer), T47D (Her2 and Ras negative breast cancer), DU-145 and, PC-3(prostate cancers) cells were grown in either DMEM or RPMI mediums with10% fetal bovine serum (FBS). Immortalized, non-transformed, Chinesehamster ovary (CHO) cells were grown in DMEM/F-12+10% FBS. HCT-116(colon cancer), UACC-62 (stable melanoma line with B-Raf and, many otheroncogenic mutations), HOP62 (lung cancer), and NCI-H23 (lung cancer)cells were obtained from NCI-60 purchased by the Biological Service Unitof our Institute. Low passage (less than 10) primary melanoma cells withB-Raf mutation (A2185, A2352, A2024), and immortalized, non-transformedbreast cells (HB2), were used. Established melanoma cell lines carryingB-Raf mutation (A2577, A2600, LOXIMVI) were from ATCC and A2058 cellswere also used. All these melanoma cells were grown in RPMI supplementedwith 10% FBS; and the HB2 cells were grown in the same mediumsupplemented with 10 μg/ml insulin and 0.5 μg/ml hydrocortisone.Immortalized, non-transformed breast MCF-10A cells (ATCC) were grown inDMEM/F12 with 5% donor horse serum, 20 ng/ml EGF, 10 μg/ml insulin, 0.5μg/ml hydrocortisone, and 100 ng/ml cholera toxin (all reagents fromSigma, Israel).

Reagents and Antibodies:

Tetradecanoyl phorbol acetate (TPA), EGF, Avidin-FITC andTGF-diamino-2-phenylindole (DAPI), 3,3′-Diaminobenzidine (DAB) wereobtained from Sigma (St Louis, Mich.). Anti general Elk1 (gElk1) andgRSK1 Abs were from Santa Cruz Biotechnology (CA, USA). Anti pElk-1(Ser383), PARP-1, pAKT (Ser473) and pRSK (Ser381) Abs were from CellSignaling Technology (Beverly, Mass., USA). Anti Imp7 Ab was from Abnova(Taipei, Taiwan). Anti pERK (pTEY-ERK), gERK, gAKT, gp38, pp38 (TGY),gJNK and pJNK (TPY) Abs were from Sigma (Rehovot, Israel). Polyclonaland monoclonal anti phospho SPS-ERK Abs were produced in the BiologicalService Unit of the Weizmann Institute of Science (Rehovot, Israel).Secondary fluorescent Ab conjugates were from Jackson Immunoresearch(West Grove, Pa.). Secondary Ab conjugated to peroxidase (Simple StainedMax PO) was from Nichirei Biosciences (Japan).

Fluorescence Microscopy:

Cells were fixed in 3% paraformaldehyde in PBS (20 min, 23° C.), andthen permeabilized and blocked with 0.2% Triton X-100 in PBS-BSA (2%)for 20 min at 23° C. The fixed cells were sequentially incubated withappropriated Abs, (diluted in 10 μg/ml BSA/PBS) for 1 h, followed byeither Cy-2 or rhodamine-conjugated secondary Abs and DAPI (diluted inBSA/PBS, 1:200) for 1 h. To follow the subcellular localization of thebiotin-conjugated peptides, cells were incubated with Avidin-FITC(diluted in BSA/PBS 1:400) and DAPI. Slides were analyzed andphotographed by a fluorescent microscope (Nikon, Japan, 600×magnification).

Western Blotting and Coimmunoprecipitation:

(i) Preparation of cellular extracts for Western Blotting: Cells wererinsed twice with ice-cold PBS and scraped intoRadio-immunoprecipitation assay buffer (RIPA; 137 mM NaCl, 20 mM Tris(pH 7.4), 10% glycerol, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 2mM EDTA, 1 mM PMSF, and 20 μM leupeptin). The extracts were centrifuged(20,000×g, 15 min, 4° C.), and the supernatants were further analyzed byWestern blotting. The blots were developed with horseradishperoxidase-conjugated anti-mouse or anti-rabbit Abs. (ii)Coimmunoprecipitation: Cells were rinsed twice with ice-cold PBS andscraped into buffer H (50 mM β-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1mM EDTA, 1 mM dithiothreitol, 0.1 mM sodium vanadate, 1 mM benzamidine,10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin). The extractswere sonicated (50 W, 2×7 sec), and centrifuged (15,000×g, 15 min, 4°C.). Cellular extracts were incubated overnight with the appropriate Abspreconjugated to A/G beads (1 h, 23° C.). Subsequently, the beads werewashed ×3 with coimmunoprecipitation washing buffer (20 mM HEPES pH 7.4,2 mM MgCl₂, 2 mM EGTA, 150 mM NaCl and 0.1% Triton-X-100) and once withPBS, and subjected to Western blot analysis.

Morphology and Apoptosis (TUNEL) Assays:

Cells were seeded on glass cover slips in a 12-wells plate at 25%confluence with medium containing 1% of FCS. Peptides (EPE or Scr),DMSO, or U0126 in a final concentration of 10 μM, were added after 4 h(considered as time “0”). TUNEL staining was performed at 24 h after thetreatment according to manufacturer instruction (Roche Applied Science,Nutley, N.J., USA). Briefly, the medium was removed and cells were fixedwith 3% of paraformaldehyde (1.5 h, 23° C.). Cells were rinsed twicewith PBS and permeabilized with 0.1% Triton X-100 and 0.1% sodiumcitrate for 2 min on ice. Cells were rinsed twice with PBS and 28 μl ofTUNEL reaction mixture (25 μl of TUNEL label, 2.5 μl of TUNEL enzyme and0.1 mg/ml of DAPI) was added directly on top of the slide, cells wereincubated for 16 h in humid box at 37° C. Cover slips were rinsed threetimes with PBS and mounted on microscope slides. The slides were driedand then subjected to image acquisition by a florescence microscope. Formorphology assay, the cells were seeded at approximately 25% confluencein 6 cm plates with medium containing 1% FCS. Peptides (EPE and Scr) orDMSO or U0126 in a final concentration of 10 μM, were added after 4 h(considered to be time 0), 24 h and 48 h. Images were obtained by alight microscope (Olympus BX51) after 72 h.

Proliferation Assay:

All cells, except of MCF-10A, were seeded into 12-well cell plates in 1%FBS medium. MCF-10A cells were seeded in their complete medium diluted 5fold. DMSO, Scr peptide, EPE peptide, or U0126 (final concentration of10 μM each) were added to the appropriated wells four hours later. Everyday medium was changed to fresh one containing the same agents. Thenumber of viable cells was measured by Methylene Blue assay at 72 hafter cell seeding. Shortly, cells were fixed with 4% Formaldehyde for 2h at 23° C., washed once with 0.1M Borate Buffer pH 8.5 and stained withof 1% Methylene Blue in 0.1M Borate Buffer for 10 min. Color wasextracted by adding 0.1 M HCl for 3 h at 23° C., and examined at 595 nm.For time course experiments, viable cells were measured at 0, 24, 48, 72and 96 h after cell seeding. For dose response experiments we treadedthe cells with 0.1, 1, 3, 10, or 30 μM of peptides for 72 h, asdescribed above.

Preparation and Proliferation of Inhibitors Resistant Melanoma Cells:

A2352 cells were supplemented with either PLX4032 (B-Raf inhibitor, 1μM), or U0126 (MEK inhibitor, 10 μM) for 45 days. Cells that survivedthis inhibitory pressure were treated with the following agents: DMSO,Scr peptide, EPE peptide, U0126 (the final concentration of 10 μM each),PLX-4032 (1 μM), Wortmannin (PI3K inhibitor, 0.5 μM), or Taxol (25ng/ml), and subjected to proliferation assay as described above.

Animal Studies:

All animal experiments were approved by the Animal Care and UseCommittee of the Weizmann Institute of Science (Rehovot, Israel). FemaleCD-1 nude mice (Harlan), 5-6 weeks of age, were inoculated s.c. into theflank region with 2×10⁶ MDA-MB-231, LOXIMVI, or HCT-116 cells in 150 μlPBS. Female SCID mice (Harlan), 5-6 weeks of age, were inoculated s.c.into the flank region with 10⁷ A2352 cells in 150 μl mixture of PBS withmatrigel (2:1). Tumors were allowed to develop to the size of 5-6.5 mmin diameter (50˜110 mm³ volume) and then the animals were randomlyallocated to different treatment groups. The peptides (100 mM stock inDMSO), were diluted to the necessary concentration in PBS and boiled for5 minutes. Then, DMSO, Scr or EPE peptides were administered by i.v.injection into the tail vein (150 μl/mouse, 3 times a week). Tumordimensions were measured with a digital sliding caliper. Tumor volumewas calculated using the formula: V=D₁×D₂×D₃×π/6, where D₁, D₂,D₃—represent the three mutually orthogonal growth diameters. To assessany signs of systemic toxicity, body weight was monitored, and recordedat the end of the experiment.

Histology and Immunohistochemistry:

Tumor xenografts, lungs, livers, kidneys and hearts of animals fromdifferent treatment groups were removed and subjected for histologicalanalysis by staining of 5 μm paraffin embedded tissue slides with H&E,and examination by light microscope. MDA-MB-231 and LOXIMVI tumorxenografts were subjected to immunohistochemical analysis using αgERKAbs. Briefly, paraffin embedded blocks of tumors after differenttreatments were cut at 4 μm thickness and stained with ERK Abs, followedby second antibodies conjugated to peroxidase and DAB staining.Representative fields of each specimen were photographed at ×20 and ×40magnifications.

Statistical Analysis:

Digital images were processed with Adobe Photoshop 7.0 software. Thestatistical differences were analyzed using two-tailed t-Students test.

RESULTS

The EPE-Peptide Inhibits ERK's Translocation:

The nuclear translocation of ERK is a key step in mediating cellularproliferation, while having only minor influence on other cellularprocesses. It was found that the stimulated translocation of ERKrequires the binding of its phosphorylated NTS with Imp7. To preventthis interaction, the present inventors used an NTS-derived peptide(GQLNHILGILGSPSQED, SPS—SEQ ID NO: 2) that could compete with thebinding. To be effective, the peptide would need to rapidly penetratethrough the cell membrane and remain in the cytoplasm for a certainamount of time. To reach this goal, the present inventors examined twoknown ways to allow peptide penetration: modified viral TAT sequence²²or myristic acid (Myr;²³), both in the N-terminus of the peptide. Usingbiotinylated peptides with each of the leaders, it was found that bothof them induced an efficient penetration into HeLa cells, but the Myrpeptide remained in the cytoplasm longer than the peptide with the TATsequence (FIGS. 7A-B).

The present inventors then undertook to examine the effect of thepeptide on the nuclear translocation of ERK1/2. Treatment of HeLa cellswith the SPS peptide prevented TPA-induced nuclear translocation of ERK,which was similar to the inhibition by the MEK inhibitor U0126 (FIG.1A). Next, the efficacy of the peptide was compared to similar peptidesin which the SPS motif was replaced with either phosphomimetic (EPE) ornonphosphorylatable (APA) residues. The inhibitory effect of the EPEpeptide was stronger than that of the other two (FIGS. 8A-B), probablybecause the EPE peptide better mimics the Imp7-bound ERK; The study wastherefore continued with this peptide only. Repetition of theexperiments with T47D, MDA-MB-231, A2352 cells and two immortalizednon-transformed cells: melanocytes (NHEM-Ad) and breast (HB2) revealed asimilar effect (FIGS. 1A-F), pointing to the generality of the effect.The same trend of inhibition was observed with subcellular fractionationas well (FIGS. 12A-D). No significant differences between the inhibitionof ERK1 and ERK2 were observed (FIGS. 13A-C), In addition, the effectwas specific to ERK, as the peptide affected neither the translocationof other MAPKs (FIGS. 9A-B), nor that of AKT (data not shown).

Molecular Effects of the EPE Peptide:

Next, the present inventors undertook to identify the mechanism by whichthe EPE peptide prevents the nuclear translocation of ERK. As expectedfrom the origin of the EPE-peptide, it was found that its addition toHeLa cells indeed prevented the interaction of Imp7 with ERK whenexamined by coimmunoprecipitation with anti Imp7 Abs (FIG. 2A). Theeffect of the peptide on the intracellular signaling of four distinctcell lines was examined. As expected, no effect of the peptide oncytoplasmic activities was detected, including either activatory ERK-TEYphosphorylation or the downstream activity of RSK (FIG. 2B). Moreover,even the NTS phosphorylation by CK2, which occurs in the cytosol, wasonly slightly affected despite the consensus CK2-phosphorylation sitewithin the peptide. This lack of effect may suggest that the bindingsites of CK2 and Imp7 to the NTS are not identical, and strongly supportthe specificity of the peptide to ERK-Imp7 interaction. Importantly, thepeptide had no effect on the basal or stimulated phosphorylation of AKT,either shortly after stimulation, or in longer time periods aftertreatment (FIGS. 2E,F), indicating that the negative feedback loops ofthe cells were not affected by the peptide. Finally, although thepeptide had no cytoplasmic effects, it did inhibit the phosphorylationof the transcription factor Elk1, which is a nuclear target of ERK1/2(FIG. 2B), the phosphorylation of c-Myc (FIG. 2C) and, to a lesserextent, the expression and phosphorylation of c-Fos (FIGS. 14A-B). Thiseffect on nuclear targets varied among the cell lines, (20-45% in Elk1,FIG. 2D), and was not so pronounced for c-Fos, probably due to theinvolvement of other, ERK1/2-independent, signaling components in somecells.

The EPE Peptide Effects in Cultured Cells:

Given that the nuclear activity of ERK1/2 is critical for cellproliferation, the present inventors then examined the effect of the EPEpeptide on proliferation/survival of different cancer-derived andimmortalized cell lines. First, the optimal administration conditions ofthe EPE peptide (in which it presented the maximal effect on HeLa andT47D cells compared to a scrambled (Scr) peptide control) was found tobe 10 μM, administered every 24 h in fresh medium (FIGS. 9A-B). Next,the present inventors examined the effect of the peptide onproliferation of different cell lines measured 72 h after peptideadministration (FIG. 3A). Interestingly, the response of the differentcells to the peptide can be categorized into four types. The first onewas a profound reduction in cell number, which was observed in melanomacells with oncogenic B-Raf (B-Raf melanoma). In the second group,including breast, prostate and cervical cancer-derived cells, thepeptide prevented cell growth, but did not reduce the number of initialcells. The third group that included other melanomas, prostate and lungcancer-derived cells presented a small decrease in cell growth ascompared to a peptide control, and a fourth group that includedimmortalized, non-transformed cells, did not respond to the peptide atall. Further comparison between the effects of EPE peptide and PLX4032on the viability of some of the cell lines (FIG. 3B), revealed that innon-transformed melanocytes (NHEM-Ad) the EPE peptide does not have anysignificant effect despite the strong inhibitory effect of PLX4032. Onthe other hand, the EPE peptide was able to reduce the viability ofN-Ras transformed melanoma cells LOXIMVI that are not sensitive toPLX4032 as was previously reported. In all other transformed cell linesexamined, the effect of the EPE-peptide was at least as good, or evenbetter, than that of PLX4032. Together, these results demonstrate thesuperior effects of the EPE peptide in treating various cancers withoutaffecting the non-transformed cells.

EPE Peptide Effect on Resistant Melanoma Cells:

The major problem with the use of B-Raf and MEK inhibitors in the clinicis the development of drug-resistance after 6 to 8 months. Since thepoint of influence of the EPE peptide is downstream to the other twodrugs, the present inventors examined whether it might affect melanomacells resistant to the Raf and MEK inhibitors. For this purpose PLX-4032and U0126 resistant cells were generated by adding the inhibitor to theA2352 melanoma line for 6 weeks. The surviving cells proliferated slowerin the presence of the inhibitors, but regained normal growth when theinhibitors were removed. Using these cells, it was found that the EPEpeptide was able to reduce cell growth, although this effect was not asimpressive as observed in the non-resistant ones (FIG. 4A).

Similar effects were seen in two low-passage melanoma cells fromvemurafenib-resistant patients (FIG. 4B). As expected, the EPE peptidereduced the amount of nuclear ERK1/2 both before and after stimulation(FIGS. 15A-B). No significant effects of the peptides were detected onthe activation of AKT were detected (data not shown), indicating thatthe effects is not through the cytosolic negative feedback loops. Thus,the present results indicate that the resistant melanoma cells arehighly responsive to the EPE peptide.

Since inhibition of the MAPK cascade often results in activation ofnegative feedback loops and stimulation of the PI3K/AKT pathway, thispathway was examined in the resistant cells as well. The resultsdemonstrate no significant change in the influence of the PI3Kinhibitor, indicating that the resistance was probably not due to thePI3K, and the EPE peptide operated via a distinct pathway. It was alsodemonstrated that the effect is not due to a change in the multidrugresistance system, as it was found that Taxol was as effective innon-resistant, as resistant cells. Thus, the present results stronglyindicate that the EPE peptide is able to affect the resistant cells viainhibition of downstream machinery.

The EPE Peptide Induces Apoptosis of Melanoma:

The effect of EPE peptide on cell morphology was visualized using lightmicroscopy. While no effect was observed in most cell lines examined,the peptide did change the appearance of B-Raf melanoma cells byinducing cell-break already 24 hours after treatment (FIG. 5A). Asimilar appearance, albeit a weaker one, was observed with the MEKinhibitor U0126 as well, indicating that this effect is likely to beMEK/ERK-dependent. This change of morphology resembled cell death, whichwas previously reported to occur upon inhibition of the ERK cascade inB-Raf melanoma. Indeed, using TUNEL (FIGS. 5B,C) or PARP-1 (FIG. 5D) asmarker for apoptosis, it was found that the morphology change correlatedwith an enhanced apoptosis. This apoptotic effect was specific to B-Rafmutated melanomas and, in these cells, was as strong as the apoptosisinduced by Taxol. No apoptosis was detected in the other cell linesexamined, despite their clear ability to undergo a Taxol or H₂O₂-inducedcell death.

The EPE Peptide Effect on Cancer Xenografts:

The present inventors then examined the effect of the EPE peptide on thegrowth of tumors in xenograft models (FIG. 6A). For this purpose, thetumors were allowed to grow to a size of ˜60 mm³ and only then thepeptide was systemically administrated by injecting it in a properformulation into the tail vein of the mice. Using such xenograft modelsin nude mice, dose dependent inhibition of the growth of MDA-MB-231,LOXIMVI was noted, and to some extent, also on HCT-116. Remarkably, aneven stronger effect of the peptide was seen with a xenograft of the lowpassage A2352 B-Raf melanoma in SCID mice. In this model, the peptidecompletely irradiated the melanoma within 2 weeks of tail veinadministration. None of the animal treated exhibited any significantchange in weight, organ morphology or other toxicity-related effects.Moreover, the treatment did not affect the size or structure of thekidneys, livers and hearts that were inspected at the end of theexperiment. Interestingly, the structure of the lungs was not affectedas well, although metastatic foci lungs in vehicle- and Scrpeptide-treated were found, but not EPE peptide-treated mice (notshown).

In order to verify that the EPE peptide indeed operated by preventingthe nuclear translocation, sections of xenograft tumors were excisedfrom the treated animals at the end of the experiments, and stained withanti ERK antibody (Ab). As expected, it was found that EPE peptide didprevent such translocation in the treated MDA-MB-231 and LOXIMVIxenografts. In samples from the control treated xenografts, ERK wasfound all over the cells, with some preference to the nucleus, while inthe EPE peptide-treated xenografts, ERK was localized almost exclusivelyin the cytoplasm (FIGS. 10A-B). These findings support the notion thatthe cytoplasmatic retention of ERK is the cause for the specific effectof the EPE peptide. Therefore, the prevention of the nucleartranslocation of ERK1/2, which do not affect the cytoplasmic activity ofthe cascade, may serve as a good tool to prevent cancer growth, withless side-effects than the currently used inhibitors of the ERK1/2cascade.

The major problem with the use of B-Raf and MEK inhibitors in the clinicis the development of resistance after 6 to 8 months, which results intumor and metastasis recurrence. In order to study the recurrence of thedisease after EPE peptide in comparison to PLX4032 treatment, micebearing A2352 xenografts were treated with both reagents. Bothtreatments were proven beneficial in reducing the size of the initial˜80 mm3 tumors. Treatment of the EPE peptide resulted in a completedisappearance of the tumors of all mice within 10-23 days (FIG. 6B),while the PLX4032 treatment resulted in a complete disappearance inthree mice (13-23 days after treatment), and two mice with small tumors.Following last treatment administration mice were kept for furtherfollow up and evaluation of melanoma condition for up to 11 weeks. Noneof the EPE-peptide-treated mouse (N=7) showed any tumor recurrence, andall of them, as well as 5 other animals in a repeating experiment,remained healthy up to 11 weeks after treatment. On the other hand, asexpected, in some (3 out of 5) of the PLX4032-treated mice, the tumordid recur, and one of them appeared to develop resistance within 13 daysof treatment and exhibited a massive tumor growth thereafter. Theseresults indicate that the EPE peptide treatment may prevent resistanceand tumor recurrence better than that of PLX4032.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

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What is claimed is:
 1. An isolated peptide attached to a cellpenetrating agent, wherein the peptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2-3, 6 and
 7. 2. Theisolated peptide of claim 1, being 17 amino acids long.
 3. The isolatedpeptide of claim 1, wherein said cell penetrating agent comprisesmyristic acid.
 4. A pharmaceutical composition comprising the peptide ofclaim 1 as the active agent and a pharmaceutically acceptable carrier.